Immunoassays have been used for decades as a means to assay for the qualitative and quantitative presence of antigens or antibodies in a sample. Immunoassays have many practical uses, including the diagnosis of certain diseases by detecting antigens that are characteristic of the disease and the screening of blood or other fluids by detecting harmful substances or organisms, or antibodies associated with the presence of the harmful substances or organisms. For example, screening of blood or other fluids for autoantibodies to DNA can be used in the diagnosis of autoimmune diseases.
One of the most common immunoassay techniques uses a solid phase matrix to which either an antigen or antibody is bound. For example, typical ELISA systems use plastic (polystyrene) 96-well plates (Microtiter plates) that have been adapted and/or modified to provide optimal binding of the antigenic substance or antibodies.
Various methods are known for attaching molecules to surfaces such as ELISA plates. These methods include attaching the molecules to the surface of derivatized surfaces by covalent binding or coating surfaces with molecules dissolved in a solvent so that the molecules adhere to the surface when the solvent evaporates.
These methods have various disadvantages relating to reproducibility, sensitivity and quantification. In a solid phase immunoassay, it is important that the attached antigen or antibody be present on the surface in an orientation so that the specific portion of the molecule that participates in ligand/receptor binding is exposed and so that the binding site is not altered or distorted. In other words, it is important that the features that make a particular molecule useful in an immunoassay not be destroyed in the process of attaching the molecule to a surface. Moreover, to insure reproducibility, the attached antigen or antibody should resistant to damage, alteration or removal during the typical harsh conditions of an immunoassay.
When molecules are attached to a derivatized surface by covalent binding, there is likelihood that chemical reactions occurring during the binding event will also alter the receptor/ligand-binding site. Furthermore, the molecules might be attached to the surface in an orientation such that the specific receptor/ligand-binding sites are not accessible. This reduces the sensitivity of the immunoassay.
When a molecule is attached to a surface by methods of coating and solvent evaporation, there is a difficulty in assuring that the molecules are bound to the surface in a reproducible manner. Moreover, if an antigen or antibody is coated onto a surface of untreated plastic, which tends to be hydrophobic, the antigen or antibody may, depending on the particular solute and surface being used, orient itself so that the hydrophobic portion of the antigen or antibody adheres to the hydrophobic plastic surface. There is then a likelihood that the receptor/ligand binding portion of the antigen or antibody will be obscured, resulting in reduced sensitivity.
A further problem with typical methods of attaching antigens and antibodies to pre-formed surfaces is that these methods tend to be expensive and labor intensive.
ELISA-based testing for autoantibodies to DNA is typically performed to assess for autoimmunity. A generic name for this type of testing is “ANA”, which stands for anti-nuclear-antibodies. The present invention demonstrates that autoantibodies to DNA embedded in polystyrene are easily detectable in patients with known ANA positive activity. The benefits of using embedded DNA are manifold and have advantages over the traditional methods of ELISA-based ANA testing. First, the embedded DNA will not fall off the plastic as it may when using the conventional “coating” of plastic which relies on non-specific (electrostatic) adherence to the ELISA plate wells. Thus, there can be standard concentrations of DNA embedded into the plastic and this will not change regardless of who performs the test and where the testing is preformed. Second, the embedded DNA antigens will have an indefinite shelf life. Third, by mixing the DNA with the plastic stock, there is no need for the expensive technician time that is required to “coat” the plate wells. Fourth, if desirable, the DNA embedded wells can be recycled by adding low pH to remove the bound antibody. To quantify the level of antibody in blood, one must have a reproducible standard assay since there is a correlation between the number of antigen molecules on the solid support and the number of bound antibody molecules resulting from sample application.
In another aspect of the present invention, a polynucleic acid with a specific sequence may be embedded into a solid material for use in a hybridization assay to detect complementary strands of polynucleic acid, such as viral or bacterial DNA or RNA. The use of embedded DNA for this purpose has the same advantages over conventional hybridization assays as it does in immunoassays, including greater reliability, improved shelf life, lower cost of manufacture and reusability.